CN110484585A - A kind of method preparing active peptides from Gorgon fruit albumen powder and active peptides and application - Google Patents
A kind of method preparing active peptides from Gorgon fruit albumen powder and active peptides and application Download PDFInfo
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- CN110484585A CN110484585A CN201910892968.2A CN201910892968A CN110484585A CN 110484585 A CN110484585 A CN 110484585A CN 201910892968 A CN201910892968 A CN 201910892968A CN 110484585 A CN110484585 A CN 110484585A
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- gorgon fruit
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
Abstract
The present invention relates to a kind of method that active peptides are prepared from Gorgon fruit albumen powder and active peptides and applications.The method includes the steps (1) deionized waters to extract, step (2) alkali carries take, the sour extraction of step (3), step (4) Tris-HCl extraction, step (5) alcohol extracting, step (6) enzymatic hydrolysis, Gorgon fruit active peptides obtained are used to prepare among food, health food and special medicine purposes formula food.Under this process condition, Gorgon fruit protein hydrolysate degree is up to 45.7% to the present invention, and polypeptide yield 37.9%, the hydroxyl radical free radical clearance rate of hydrolysate is 65.1%, and ultra-oxygen anion free radical clearance rate is 45%.The simple in production process operation, mild condition, good to Gorgon fruit proteolysis situation, antioxidant activity polypeptide yield is high, and Degree of Enzymatic Hydrolysis is controllable, provides effective reference for the deep processing and comprehensive utilization of Gorgon fruit albumen.
Description
Technical field
The invention belongs to food processing technology field, be related to a kind of method that active peptides are prepared from Gorgon fruit albumen powder and
Active peptides and application.
Background technique
Biologically active peptide refers to that protein passes through the peptide matters with biological activity that degradation obtains, molecule knot
Structure complexity is different, can be as simple as only that there are two the dipeptides of amino acid composition, is also possible to the macromolecular of complicated annular
Polypeptide, the special physiological function that there is polypeptide the amino acid of protein and composition polypeptide all not necessarily to have.Moreover, very much
Polypeptide with high bioactivity might not be entirely to be made of essential amino acid, so that as the ammonia Gorgon fruit albumen powder
Base acid composition is not that very ideal, essential amino acid composition is not very comprehensive, is typically considered non-prime egg
The deep processing of some protein resources of white matter, recycling provide outlet.Therefore, polypeptide is more open using space, answers
It is higher with being worth.
There is a large amount of research to confirm that biologically active peptide has the function of multiple biological activities at present, such as: adjusting is exempted from
Epidemic disease, the content for reducing Blood Cholesterol, the formation for inhibiting thrombus etc., biologically active peptide has become the day of many drugs
Right source.Extraction, which prepares biologically active peptide, from food protein or raw-food material becomes the development trend of biologically active peptide preparation,
Have many advantages, such as that safe source is reliable, toxic side effect is small, low in cost, the hot spot of researcher's research is increasingly becoming, by blueness
It looks at.With the development of economy, people's lives rhythm is increasingly accelerated, and various " rich people's diseases " occur again and again, and most people is being located
In or be once in the state of inferior health, excessive life stress makes the physiological dysfunction of people, takes the photograph only by food
Enter the needs that human body has been not achieved in required nutriment, and powerful pressure causes human body itself to synthesize functional polypeptide
Ability weaken significantly, therefore most of people starts to focus on supplementing the normal need of human body by taking in exogenous peptide
It wants.
It is drug substances raw material again that it is food that Gorgon fruit, which belongs to, and the polypeptide contained, which has, nourishes strong, tonifying middle-Jiao and Qi, benefit
The effects of kidney controlling nocturnal emission with astringent drugs, can reduce human-body fatigue sense, remove the physiological activity such as interior free yl, make its great development prospect, can be with
It is widely used in food and related fields.
Currently, the research of Gorgon fruit polypeptide to sum up there is a problem in that: (1) have focused largely on high yield pulp1 Gorgon fruit peptide institute
It with the research in the screening of protease, and is generally that Gorgon fruit albumen powder is hydrolyzed using single enzyme；(2) Gorgon fruit protein
Configuration space is smaller, close structure, and has stronger hydrophobicity, is unfavorable for protease and it is hydrolyzed.
Summary of the invention
In order to solve the above-mentioned technical problem, the object of the invention is that providing one kind prepares active peptides from Gorgon fruit albumen powder
Method.In order to achieve the object of the present invention, inventor is studied by a large number of experiments, is finally obtained following technical solution:
A method of active peptides being prepared from Gorgon fruit albumen powder, are included the following steps:
(1) deionized water is extracted: being weighed Gorgon fruit albumen, in 1:10-30 ratio extracting in water, is extracted 2-3 at 4-8 DEG C
Secondary, 3-5h/ times, extracting solution is centrifuged 10-15min, and supernatant is Gorgon fruit polypeptide crude extract A；
(2) alkali carries take: weighing step (1) crude extract A in 1:10-20 ratio extracting in water and pH is adjusted to 9.0, in 4-8
DEG C extract 2-4 time, 3-5h/ times, extracting solution centrifugation 10-15min, supernatant is Gorgon fruit polypeptide crude extract B；
(3) acid extracts: crude extract B described in step (2) is weighed, 300-500mL distilled water is added, pH is adjusted to 3.0-5.0,
It is extracted in 4-8 DEG C 2-4 times, 3-5h/ times, extracting solution is centrifuged 10-15min, and supernatant is Gorgon fruit polypeptide crude extract C；
(4) Tris-HCl is extracted: weigh crude extract C described in step (3), with the Tris-HCl solution of 5-15mmol/L into
Row extracts, and pH is adjusted to 6-8, extracts in 4-8 DEG C 2-4 times, 3-5h/ times, extracting solution is centrifuged 10-15min, and supernatant is Gorgon fruit
Polypeptide crude extract D；
(5) alcohol extracting: weighing crude extract D described in step (4), adds 40-70% ethanol solution to carry out separation and Extraction, in 4-
8 DEG C are extracted 2-4 times, and 3-5h/ times, extracting solution is centrifuged 10-15min, and supernatant is Gorgon fruit polypeptide crude extract E；
(6) it digests: weighing crude extract E described in step (5), alkali protease and neutral proteinase complex enzyme, pH is added
Value 5-8, solid-to-liquid ratio 1:10-20, enzyme concentration 1-3%, hydrolysis time 1-3h, 40-65 DEG C of temperature；Alkali protease and neutral protein
Enzyme ratio is 1-3:1-5；Then enzymolysis liquid is concentrated, then is freeze-dried, encapsulated.
Preferably, the revolving speed of centrifugation described in each step is 4000-6000r/min.
Preferably, pH is adjusted to 9.0 with the NaOH of 1-5mol/L in step (2).
Preferably, pH is adjusted to 3.0-5.0 with the acetic acid of 1-5mol/L in step (3).
Preferably, alkali protease and neutral proteinase ratio are 1-3:1-5.
Preferably, step (6) includes: to weigh crude extract E described in step (5), and alkali protease and neutral protein is added
Enzyme ratio 1.5:1, pH value 7.5, solid-to-liquid ratio 1:10, enzyme concentration 2.3%, hydrolysis time 2.5h, 60.5 DEG C of temperature.
The present invention also provides Gorgon fruit active peptides prepared by the above method.
The Gorgon fruit active peptides of the invention can be used for preparing food, for example, health food and special medicine purposes
Formula food.
The invention has the advantages that:
Compared with prior art, under this process condition, Gorgon fruit protein hydrolysate degree is up to 45.7% to the present invention, polypeptide yield
It is 37.9%, the hydroxyl radical free radical clearance rate of hydrolysate is 65.1%, and ultra-oxygen anion free radical clearance rate is 45%.The life
Production. art is easy to operate, mild condition, good to Gorgon fruit proteolysis situation, and antioxidant activity polypeptide yield is high, and Degree of Enzymatic Hydrolysis can
Control, provides effective reference for the deep processing and comprehensive utilization of Gorgon fruit albumen.
Other features and advantages of the present invention will then part of the detailed description can be specified.
Detailed description of the invention
Exemplary embodiment of the invention is described in more detail in conjunction with the accompanying drawings, it is of the invention above-mentioned and its
Its purpose, feature and advantage will be apparent.
Fig. 1 is complex enzyme for hydrolyzing single factor experiment result.
Specific embodiment
The preferred embodiment of the present invention is described in more detail below.Although the following describe preferred implementations of the invention
Mode, however, it is to be appreciated that may be realized in various forms the present invention without that should be limited by the embodiments set forth herein.
Embodiment 1
The present embodiment provides a kind of methods that active peptides are prepared from Gorgon fruit albumen powder, include the following steps:
(1) deionized water is extracted: Gorgon fruit albumen is weighed, in 1:10 ratio extracting in water, extracts 2 times at 4 DEG C, 3h/ times,
Extracting solution is centrifuged 10min (4000r/min), and supernatant is Gorgon fruit polypeptide crude extract A；
(2) alkali carries take: step (1) crude extract A are weighed, in 1:10 ratio extracting in water, with the NaOH of 1mol/L by pH tune
To 9.0, extracted in 4 DEG C 2 times, 3h/ times, extracting solution is centrifuged 10min (4000r/min), and supernatant is Gorgon fruit polypeptide crude extract
B；
(3) acid extracts: weighing crude extract B described in step (2), adds 300mL distilled water, with the acetic acid of 1mol/L by pH tune
To 3.0, extracted in 4 DEG C 2 times, 3h/ times, extracting solution is centrifuged 10min (4000r/min), and supernatant is Gorgon fruit polypeptide crude extract
C；
(4) Tris-HCl is extracted: being weighed crude extract C described in step (3), is carried out with the Tris-HCl solution of 5mmol/L
It extracts, pH is adjusted to 6, extracted in 4 DEG C 2 times, 3h/ times, extracting solution is centrifuged 105min (4000r/min), and supernatant is Gorgon fruit
Polypeptide crude extract D；
(5) alcohol extracting: weighing crude extract D described in step (4), adds 40% ethanol solution to carry out separation and Extraction, mentions in 4 DEG C
It takes 2 times, 3h/ times, extracting solution is centrifuged 10min (4000r/min), and supernatant is Gorgon fruit polypeptide crude extract E；
(6) it digests: weighing crude extract E described in step (5), alkali protease and neutral proteinase complex enzyme, pH is added
Value 5, solid-to-liquid ratio 1:10, enzyme concentration 1%, hydrolysis time 1h, 40 DEG C of temperature；Alkali protease and neutral proteinase ratio are 1:
1.5；Then enzymolysis liquid is concentrated, then is freeze-dried, encapsulated.
Embodiment 2
The present embodiment provides a kind of methods that active peptides are prepared from Gorgon fruit albumen powder, include the following steps:
(1) deionized water is extracted: Gorgon fruit albumen is weighed, in 1:20 ratio extracting in water, extracts 3 times at 5 DEG C, 4h/ times,
Extracting solution is centrifuged 12min (5000r/min), and supernatant is Gorgon fruit polypeptide crude extract A.
(2) alkali carries take: step (1) crude extract A are weighed, in 1:15 ratio extracting in water, with the NaOH of 3mol/L by pH tune
To 9.0, extracted in 5 DEG C 3 times, 4h/ times, extracting solution is centrifuged 12min (5000r/min), and supernatant is Gorgon fruit polypeptide crude extract
B。
(3) acid extracts: weighing crude extract B described in step (2), adds 400mL distilled water, with the acetic acid of 3mol/L by pH tune
To 4.0, extracted in 5 DEG C 3 times, 4h/ times, extracting solution is centrifuged 12min (5000r/min), and supernatant is Gorgon fruit polypeptide crude extract
C。
(4) Tris-HCl is extracted: being weighed crude extract C described in step (3), is carried out with the Tris-HCl solution of 10mmol/L
It extracts, pH is adjusted to 7, extracted in 5 DEG C 3 times, 4h/ times, extracting solution is centrifuged 12min (5000r/min), and supernatant is that Gorgon fruit is more
Peptide crude extract D.
(5) alcohol extracting: weighing crude extract D described in step (4), adds 60% ethanol solution to carry out separation and Extraction, mentions in 5 DEG C
It takes 3 times, 4h/ times, extracting solution is centrifuged 12min (5000r/min), and supernatant is Gorgon fruit polypeptide crude extract E.
(6) it digests: weighing crude extract E described in step (5), alkali protease and neutral proteinase complex enzyme, pH is added
Value 6, solid-to-liquid ratio 1:15, enzyme concentration 2%, hydrolysis time 2h, temperature 60 C；Alkali protease and neutral proteinase ratio are 3:2；
Then enzymolysis liquid is concentrated, then is freeze-dried, encapsulated.
Embodiment 3
The present embodiment provides a kind of methods that active peptides are prepared from Gorgon fruit albumen powder, include the following steps:
(1) deionized water is extracted: Gorgon fruit albumen is weighed, in 1:30 ratio extracting in water, extracts 3 times at 8 DEG C, 5h/ times,
Extracting solution is centrifuged 15min (6000r/min), and supernatant is Gorgon fruit polypeptide crude extract A.
(2) alkali carries take: step (1) crude extract A are weighed, in 1:20 ratio extracting in water, with the NaOH of 5mol/L by pH tune
To 9.0, extracted 4 times 5h/ times in -8 DEG C, extracting solution is centrifuged 15min (6000r/min), and supernatant is Gorgon fruit polypeptide crude extract
B。
(3) acid extracts: weighing crude extract B described in step (2), adds 500mL distilled water, with the acetic acid of 5mol/L by pH tune
To 5.0, extracted in 8 DEG C 4 times, 5h/ times, extracting solution is centrifuged 15min (6000r/min), and supernatant is Gorgon fruit polypeptide crude extract
C。
(4) Tris-HCl is extracted: being weighed crude extract C described in step (3), is carried out with the Tris-HCl solution of 15mmol/L
It extracts, pH is adjusted to 8, extracted in 8 DEG C 4 times, 5h/ times, extracting solution is centrifuged 15min (6000r/min), and supernatant is that Gorgon fruit is more
Peptide crude extract D.
(5) alcohol extracting: weighing crude extract D described in step (4), adds 70% ethanol solution to carry out separation and Extraction, mentions in 8 DEG C
It takes 4 times, 5h/ times, extracting solution is centrifuged 15min (6000r/min), and supernatant is Gorgon fruit polypeptide crude extract E.
(6) it digests: weighing crude extract E described in step (5), alkali protease and neutral proteinase ratio 1.5:1 is added,
PH value 7.5, solid-to-liquid ratio 1:10, enzyme concentration 2.3%, hydrolysis time 2.5h, then enzymolysis liquid are concentrated by 60.5 DEG C of temperature, then into
Row freeze-drying, encapsulation.
Test case 1
Single enzyme selection selects 5 for after Gorgon fruit protein solution ultrasonic treatment (power 140W, 10min, work 3s, interval 2s)
Test is hydrolyzed in kind protease.It is arranged hydrolysis solid-to-liquid ratio 1:20 (g:ml, similarly hereinafter), hydrolysis time 3h, enzyme concentration 3%,
PH value and temperature carry out under the optimum pH and optimum temperature of each enzyme.Measure Gorgon fruit protein hydrolysis degree, polypeptide yield and water
The antioxidant activity of product is solved, the results are shown in Table 1.
Table 1
| Measured value | Alkali protease | Neutral proteinase | Trypsase | Papain | Pepsin |
| Protein hydrolysis degree | 22.56 | 22.14 | 32.76 | 12.34 | 6.69 |
| Polypeptide yield | 21.28 | 18.98 | 15.54 | 9.91 | 5.15 |
| OH clearance rate | 50.12 | 45.31 | 43.12 | 31.19 | 23.05 |
| ·O2Clearance rate | 34.69 | 32.17 | 33.09 | 26.43 | 23.51 |
As shown in Table 1, alkali protease, neutral proteinase, trypsase are preferable to the hydrolysis effect of Gorgon fruit albumen, and wooden
Melon protease, pepsin are poor to the hydrolysis effect of Gorgon fruit.Alkali protease, neutral proteinase hydrolysis Gorgon fruit albumen are more simultaneously
Peptide yield is higher, and hydrolysate also has stronger antioxidant activity；And trypsase, papain, pepsin hydrolysis
Gorgon fruit polypeptide yield compared with alkali protease, neutral proteinase it is low, the antioxidant activity of hydrolysate is also poor, to sum up,
Single enzymolysis Gorgon fruit albumen effect is best for alkali protease.
Test case 2
Complex enzyme screening
It based on alkali protease, combines other 4 kinds of enzymes with alkali protease, ultrasound is carried out to Gorgon fruit protein solution
After processing (power 140W, 10min, work 3s, interval 2s), review enzyme hydrolysis experiment is carried out, setting hydrolysis solid-to-liquid ratio is
1:20, hydrolysis time 3h, enzyme concentration 3%, review enzyme ratio are that 1:1, pH value and temperature are average in the optimum pH of each complex enzyme
It is carried out under value and optimum temperature average value.Measure the antioxidant activity of Gorgon fruit protein hydrolysis degree, polypeptide yield and hydrolysate, knot
Fruit is as shown in table 2.
Table 2
As shown in Table 2, the alkali protease degree of hydrolysis with neutral proteinase, trypsase composite hydrolysis Gorgon fruit albumen respectively
Larger, the antioxidant activity of polypeptide yield and hydrolysate is very high and papain, pepsin composite hydrolysis Gorgon fruit albumen
Degree of hydrolysis, polypeptide yield, the antioxidant activity of hydrolysate it is all relatively low.It is comprehensively compared, selects alkali protease+neutrality
Protease is used as most suitable compound enzyme system.
Test case 3
Complex enzyme for hydrolyzing single factor experiment
Initial reaction condition are as follows: pH value 7.5, solid-to-liquid ratio 1:20, enzyme concentration 3%, hydrolysis time 3h, temperature 50 C,
Enzyme ratio (alkali protease: neutral proteinase, similarly hereinafter) 1:1, each single factor test optimum value can be fixed to subsequent list after being determined
In Factor Experiment, as a result as shown in Figure 1.
It can determine that best complex enzyme hydrolysis process parameter is respectively as follows: pH value 7.5 by Fig. 1 result, solid-to-liquid ratio 1:10 is enzyme
Amount 2.3%, hydrolysis time 2.5h, temperature 60.5, enzyme ratio 1.5:1.
Various embodiments of the present invention are described above, above description is exemplary, and non-exclusive, and
It is not limited to disclosed each embodiment.Without departing from the scope and spirit of illustrated each embodiment, for this skill
Many modifications and changes are obvious for the those of ordinary skill in art field.
Claims (9)
1. a kind of method for preparing active peptides from Gorgon fruit albumen powder, which comprises the following steps:
(1) deionized water is extracted: being weighed Gorgon fruit albumen, in 1:10-30 ratio extracting in water, is extracted 2-3 times at 4-8 DEG C, 3-
5h/ times, extracting solution is centrifuged 10-15min, and supernatant is Gorgon fruit polypeptide crude extract A；
(2) alkali carries take: weighing step (1) crude extract A and pH is adjusted to 9.0, is mentioned in 4-8 DEG C in 1:10-20 ratio extracting in water
It takes 2-4 times, 3-5h/ times, extracting solution is centrifuged 10-15min, and supernatant is Gorgon fruit polypeptide crude extract B；
(3) acid extracts: weighing crude extract B described in step (2), adds 300-500mL distilled water, pH is adjusted to 3.0-5.0, in 4-
8 DEG C are extracted 2-4 times, and 3-5h/ times, extracting solution is centrifuged 10-15min, and supernatant is Gorgon fruit polypeptide crude extract C；
(4) Tris-HCl is extracted: being weighed crude extract C described in step (3), is mentioned with the Tris-HCl solution of 5-15mmol/L
It takes, pH is adjusted to 6-8, extracted in 4-8 DEG C 2-4 times, 3-5h/ times, extracting solution is centrifuged 10-15min, and supernatant is Gorgon fruit polypeptide
Crude extract D；
(5) alcohol extracting: weighing crude extract D described in step (4), adds 40-70% ethanol solution to carry out separation and Extraction, in 4-8 DEG C
It extracts 2-4 times, 3-5h/ times, extracting solution is centrifuged 10-15min, and supernatant is Gorgon fruit polypeptide refining liquid E；
(6) it digests: weighing crude extract E described in step (5), alkali protease and neutral proteinase complex enzyme is added, by pH value
It is adjusted to 5-8, solid-to-liquid ratio 1:10-20, enzyme concentration 1-3%, hydrolysis time 1-3h, 40-65 DEG C of temperature；Then enzymolysis liquid is concentrated,
It is freeze-dried, is encapsulated again.
2. the method according to claim 1 for preparing active peptides from Gorgon fruit albumen powder, wherein described in each step from
The revolving speed of the heart is 4000-6000r/min.
3. the method according to claim 1 for preparing active peptides from Gorgon fruit albumen powder, wherein step uses 1- in (2)
PH is adjusted to 9.0 by the NaOH of 5mol/L.
4. the method according to claim 1 for preparing active peptides from Gorgon fruit albumen powder, wherein step uses 1- in (3)
PH is adjusted to 3.0-5.0 by the acetic acid of 5mol/L.
5. the method according to claim 1 for preparing active peptides from Gorgon fruit albumen powder, wherein alkali protease is in
Property protease ratio be 1-3:1-5.
6. the method according to claim 1 for preparing active peptides from Gorgon fruit albumen powder, wherein step (6) includes: to claim
It takes crude extract E described in step (5), is added alkali protease and neutral proteinase ratio 1.5:1, pH value 7.5, solid-to-liquid ratio 1:
10, enzyme concentration 2.3%, hydrolysis time 2.5h, 60.5 DEG C of temperature.
7. Gorgon fruit active peptides made from the method as described in any one of claim 1-6.
8. Gorgon fruit active peptides as claimed in claim 7 are preparing the application in food.
9. application according to claim 8, wherein the food is health food or special medicine purposes formula food.
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